Efficient preservation of genetic resources of the rhesus monkey (Macaca mulatta) is critical to the mission of the National Center for Research Resources. This application is aimed at the development of efficient macaque sperm cryopreservation and will aid in successful propagation of germplasm through artificial insemination and in vitro fertilization programs. Non-human primates (NHPs), especially genetically unique macaques, are important models of human disease with applications to infertility and contraception, infectious diseases and vaccine development, drug and alcohol addiction, neurological disorders, and regenerative medicine. Similar to human sperm, cryopreserved sperm from NHPs show variable cryoprotection success depending on the individual sperm donor. Semen donors are often selected based on successful cryopreservation results, however, many individual males, including rhesus males in research programs, have semen quality that fails to survive cryopreservation. This is likely to be particularly true concerning NHPs generated from embryonic stem cells, somatic cell nuclear transfer, and transgenics where propagation of a specific individual's genotype is desired. This grant analyzes some of the mechanisms that may cause poor cryoperformance and focuses on methods to uniformly cryopreserve these bimodal populations. Our central hypothesis is that an understanding of cryopreservation-associated physiological responses in sperm will lead to improvements in sperm cryosurvival. Our long-term goal is to develop an understanding of crucial mechanisms involved in cryopreservation of sperm in order to optimally preserve sperm from a variety of genotypically unique and valuable NHP males. Our approach will be to investigate the mechanisms of osmotic and oxidative cellular stress at the cell physiology level which includes membrane, cell signaling, and subcellular assessment of sperm function using fluorescence microscopy, flow cytometry, cryomicroscopy, and proteomics methods. As a team with members from UC Davis, Indiana University/Purdue University, and the University of Newcastle (Australia), we are well positioned to undertake the proposed research, because of our combined experience with fundamental cryobiology and sperm cell biology.